Abstract: In order to prepare monoclonal antibodies against Mycobacterium bovis(M.bovis),this experiment was carried out by immunizing BALB/c mice by subcutaneous injection of the prokaryotic expression of M.bovis important antigen protein MPB70.Using cell fusion technology,after 5 subclones and screening,the spleen cells of the well-immunized mice and the myeloma cells SP2/0 were subjected to cell fusion under the action of PEG3500.Two hybridoma cells secreting anti-M.bovis MPB70 protein were screened and named as Anti-B-MPB70:8 and Anti-B-MPB70:12,respectively.The indirect ELISA method was used to detect the affinity constant of the collected ascites,the titer analysis and the subclass identification.The purity of the monoclonal antibody was analyzed by SDS-PAGE test,the concentration of monoclonal antibodies was detected and the reaction characteristics of monoclonal antibodies were identified by Western blotting.The results showed that the purified MPB70 protein had a molecular mass of 20 ku and a concentration of 0.5 mg/mL.The affinity constants of the two monoclonal antibodies were 9.95×10⁹ and 9.53×10⁸,respectively;The antibody titers were 1:102 400 and 1:12 800,respectively;The antibody subclasses were IgG_2b and IgG₁,respectively,and the light chain types were κ type.The purity was greater than 90%;The concentrations were 3.0 and 2.2 mg/mL,respectively,and had good reactivity.The above results indicated that the monoclonal antibody against MPB70 protein had been successfully prepared,which could lay a foundation for the research of bovine tuberculosis pathogen and antibody detection technology.

Key words: Mycobacterium tuberculosis; MPB70 protein; monoclonal antibody